The long-term goals of this project are: l. To understand how enzyme structure controls catalytic specificity in modular polyketide synthases (PKSs), a large family of multifunctional enzymes involved in biosynthesis of diverse natural products; 2. To use this knowledge for the biosynthesis of rationally designed polyketides; and 3. To use the same information for the generation of chemical diversity through the design and construction of genetic libraries of mutant PKSs with varying catalytic specificities. The model system for the research proposed here is the deoxyerythronolide B synthase (DEBS; 10,283 aa), which is responsible for the biosynthesis of 6 deoxyerythronolide B (6-dEB), the 14 membered macrolide precursor of erythromycin. This enzyme complex is made up of 3 very large proteins and includes 29 putative active sites. Recently, it has been expressed in a heterologous host using a genetic strategy amenable to convenient mutagenesis. Substantial quantities of 6-dEB and 8,8a-deoxyoleandolide (which is identical to 6dEB except for an acetate starter unit) were isolated from the recombinant system. Using the above expression system, the specific aims for this proposal period include: 1. Construction and analysis of a series of mutants aimed at elucidating the catalytic degrees of freedom within a modular PKS, 2. Heterologous expression of a second modular PKS (furanomycin synthase; FS) using the above expression system, 3. Combinatorial expression of modules (or domains thereof) from DEBS and FS, and 4. Development of an in vitro system for structural and mechanistic studies on DEBS and its mutants. In addition to providing valuable insights into structure-function relationships within modular PKSs, the research proposed here could also have an impact on the areas of drug discovery and improvement. In particular, the results of this work will shed light upon the. biosynthetic degrees of freedom within modular PKSs. At the same time, the tools developed during these studies should be applicable for the manipulation and analysis of other modular PKSs from actinomycetes with poorly developed genetic tools. In turn, these conceptual and technological advances could lead to the development of genetic engineering strategies for the generation of chemical diversity within a medicinally important class of compounds.